A phase I/II study of trichosanthin treatment of HIV disease

Trichosanthin, a ribosomal inhibitor protein, blocks HIV replication in lymphocytes and macrophages. This agent was used to treat 51 patients with advanced HIV disease in a dose-escalation study in which three injections were administered over a 9-21-day period in a dose range of 10-30 micrograms/kg per injection. The maximum tolerated dose was estimated to be 30 micrograms/kg. Reversible but severe fatigue and myalgias were the major dose-limiting side-effects; mild leucocytosis and elevations in serum transaminases were noted and were reversible. Non-dose-related reversible mental status changes were seen in six patients and were considered to be associated with the drug. This was usually manifest as dementia, but progressed to coma in two patients. This reversed, but the sequelae resulted in death in one patient. Decreases in serum p24 antigen levels were noted 1 month after the first infusion in 10 of 18 patients who entered the study with elevated levels; one converted to negative. Values usually remained low to the end of the study period (2 months). In those patients with CD4+ cell levels greater than 50 x 10(6) cells/l significant decreases in sedimentation rate and increases in CD4+ cell numbers were also noted. These changes were found at all dose levels but only in patients receiving three infusions.     

A prospective study assessing the efficacy of abdominal computed tomography scan without bowel preparation in diagnosing intestinal wall and luminal lesions in patients presenting to the emergency room with abdominal complaints

AIM: To evaluate the positive predictive value of abdominal non-prepared computed tomography (CT) for diagnosing intestinal lumen or wall lesions in patients presenting to the emergency room (ER) with abdominal complaints. METHODS: For 1-year we prospectively evaluated all ER patients hospitalized after abdominal CT scan detected either intraluminal or intestinal wall lesions. These patients underwent colonoscopy serving as gold standard. Patients with prior abdominal pathology or CT findings of appendicitis or diverticulitis were excluded. RESULTS: Five hundred and sixty-eight abdominopelvic CT scans were performed in the ER, 96 had positive colonic findings. Sixty-two patients were excluded, 46 because of diverticulitis or appendicitis, 16 because of prior abdominal pathology. Of the remaining 34 patients, 14 did not undergo colonoscopy during hospitalization. Twenty eligible patients were included in the study. The positive predictive value of the CT scans performed in the ER was calculated to be 45% (95% CI 25-67). CONCLUSION: CT findings correlated with colonoscopic findings only in approximately half of the cases. Relying on non-prepared CT scan findings in planning patient management and colonoscopy may lead to unnecessary diagnostic work-ups.     

Biotransformation of melatonin in human breast cancer cell lines: role of sulfotransferase 1A1

The biologically active melatonin metabolite, 6-hydroxymelatonin (6-OHMel), is conjugated to form 6-hydroxymelatonin sulfate (6-OHMelS). To elucidate the role of the sulfotransferase (SULT) enzyme 1A1, considerably expressed in normal and malignant human breast cells, we measured the formation of 6-OHMelS by ELISA in hormone-dependent MCF-7 and hormone-independent MDA-MB231 (MDA) breast cancer cell lines after stable transfection with SULT1A1. In parent MDA cells, low SULT1A1 mRNA expression was associated with moderate 6-OHMelS formation as determined after application (24 hr) of 0.1 microM 6-OHMel. As expected, overexpression of SULT1A1 in MDA cells resulted in a 2.9- and 110-fold increase in 6-OHMelS in the cytosol and cellular supernatant respectively. Furthermore, 6.3- and 115-fold increases were observed after 0.5 microM, and 12.6- and 101-fold increases after 1 microM 6-OHMel respectively. In MCF-7 cells, because of high basal SULT1A1 expression, only two- to threefold increases in 6-OHMelS were observed after transfection with the enzyme. In total, 866 and 539 pmol/mg protein 6-OHMelS were formed from 1 microM 6-OHMel in SULT1A1 overexpressing MDA and MCF-7 cells, respectively, whereas application of 1 microM melatonin produced only <1% of 6-OHMelS. Possible interactions with the SULT1A1 substrate tamoxifen (tam), an anti-estrogen applied in the therapy of breast cancer, were also studied. A concentration of 1 microM tam increased 6-OHMelS formation by approximately threefold in the presence of 1 microM melatonin or 1 microM 6-OHMel respectively. However, no alterations were detected after application of 1 microM 4-hydroxy-tamoxifen. In summary, we demonstrate the importance of SULT1A1 for the biotransformation of 6-OHMel in human breast cancer cells. Our data further suggest that tam can modulate melatonin biotransformation.     

The effect of a protein-bound polysaccharide from coriolus versicolor on immunological parameters and experimental infections in mice

Summary The influence of PSK, a protein bound polysaccharide fromCoriolus versicolor on various immunological parameters was studied. PSK was found to enhance B cell activity as measured by the spleen plaque-forming cell assay in mice, and to stimulate mouse macrophages as determined by an enhancement of carbon clearance and an increase in the phagocytosis of opsonized sheep red blood cells by peritoneal mouse macrophages in vitro. The activation of mouse macrophages by PSK appeared to correlate with the therapeutic effects of the compound. In mice made granulocytopenic with cyclophosphamide and subsequently infected with a variety of gram-negative pathogens or withCandida albicans, PSK prolonged the average survival time of the animals. The compound also led to a drastic increase in the number of animals surviving such experimental infections as compared to untreated controls. Possible mechanisms responsible for these protective effects by PSK are discussed.

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Die Wirkung eines Protein-assoziierten Polysaccharids aus Coriolus versicolor auf immunologische Parameter und experimentelle Infektionen in Mäusen

Zusammenfassung Die Wirkung von PSK, einem an Protein gebundenen Polysaccharid vonCoriolus versicolor auf verschiedene immunologische Parameter wurde untersucht. PSK führt bei Mäusen zu einer Steigerung der humoralen Immunantwort, gemessen an der Zahl der Milzzellen, die Antikörper gegen Schaferythrozyten produzieren. Außerdem ist nach PSK-Applikation eine erhöhte Makrophagenaktivität in vitro und in vivo feststellbar. Für die Messung der Makrophagenaktivität in vitro wurde die Erythrophagozytose opsonisierter Schaferythrozyten, für die Bestimmung der Aktivität des mononukleär-phagozytären Systems in vivo der Carbon Clearance Test eingesetzt. Mäuse, in denen mit Hilfe von Cyclophosphamid eine Granulozytopenie erzeugt wurde, konnten durch Applikation von PSK vor den letalen Konsequenzen experimenteller Infektionen mit einer Reihe gramnegativer Organismen und mitCandida albicans geschützt werden. In den Infektionsversuchen wurde sowohl die durchschnittliche Überlebensdauer der mit PSK behandelten Tiere verlängert als auch die Zahl der überlebenden Tiere gegenüber entsprechenden Kontrollgruppen erhöht. Diese therapeutischen Effekte scheinen am besten mit der durch PSK ausgelösten Aktivierung der mononukleären Phagozyten korrelierbar zu sein. Mögliche Mechanismen, die der beobachteten Schutzwirkung zugrunde liegen können, werden diskutiert.

     

Molecular lock regulates binding of glycine to a primitive NMDA receptor

The earliest metazoan ancestors of humans include the ctenophore Mnemiopsis leidyi. The genome of this comb jelly encodes homologs of vertebrate ionotropic glutamate receptors (iGluRs) that are distantly related to glycine-activated NMDA receptors and that bind glycine with unusually high affinity. Using ligand-binding domain (LBD) mutants for electrophysiological analysis, we demonstrate that perturbing a ctenophore-specific interdomain Arg-Glu salt bridge that is notably absent from vertebrate AMPA, kainate, and NMDA iGluRs greatly increases the rate of recovery from desensitization, while biochemical analysis reveals a large decrease in affinity for glycine. X-ray crystallographic analysis details rearrangements in the binding pocket stemming from the mutations, and molecular dynamics simulations suggest that the interdomain salt bridge acts as a steric barrier regulating ligand binding and that the free energy required to access open conformations in the glycine-bound LBD is largely responsible for differences in ligand affinity among the LBD variants.Glutamate receptor ion channels (iGluRs) are membrane proteins that mediate excitatory synaptic transmission in the brain by detecting release of the amino acid glutamate from nerve terminals (1). In combination with GluN2 subunits, which bind glutamate, NMDA subtype iGluRs use glycine as a coagonist, which binds to GluN1, GluN3A, and GluN3B subunits (2–6). NMDA receptors play key roles in synaptic plasticity and memory formation, and mutations of NMDA receptor genes underlie a diverse set of neurological and psychiatric diseases (7). Like all iGluRs, NMDA receptors are assembled from modular subunits containing amino terminal and S1S2 ligand binding domains (LBDs), which can be genetically isolated and expressed as soluble proteins for biochemical and structural analysis (4, 8–10). The LBDs of both the glutamate and glycine binding subunits are clamshell-shaped proteins of molecular mass around 30 kDa in which two lobes are connected by a hinge formed by antiparallel β-strands; in the activated state, ligands are trapped in a cavity formed when the clamshell closes. Strikingly, the volume of the ligand binding cavity for the GluN1, GluN3A, and GluN3B subunits is just large enough to accommodate glycine, whereas iGluR glutamate binding subunits have cavities that are four to five times larger and bind both glutamate and up to six or seven water molecules (4, 10–13).We recently reported the discovery of glycine-activated iGluRs from the comb jelly Mnemiopsis leidyi and the sea gooseberry Pleurobrachia bachei, candidates for earliest lineage metazoans, for which ML032222a and PbGluR3 glycine complex crystal structures reveal a salt bridge at the perimeter of the ligand binding cleft (14). This salt bridge links the upper and lower lobes of the LBD in the closed cleft glycine-bound conformation. Ctenophore iGluR subunits bind glycine with such high affinity that the ligand cannot be removed by exhaustive dialysis, suggesting an unusually stable ligand-bound closed-cleft conformation, perhaps stabilized by the interdomain salt bridge. Prior electrophysiological and crystallographic studies on vertebrate AMPA and kainate subtype iGluRs revealed that the stability of the closed cleft conformation is determined not only by contacts of the LBD with the neurotransmitter ligand but also by contacts formed between the upper and lower lobes of the clamshell assembly that occur only in the ligand-bound closed-cleft conformation (15, 16). Comparison of crystal structures of ctenophore iGluR LBDs with those of vertebrate NMDA receptor GluN1 and GluN3 subunits that also bind glycine, but for which apo proteins can be prepared without difficulty (4, 10), reveals that the salt bridge is unique to ctenophore iGluRs, further suggesting that it might underlie the high stability of the glycine complex.To investigate this, we prepared ML032222a mutant proteins and analyzed their ligand binding properties using electrophysiological, biochemical, and crystallographic techniques. To gain further insight into how these mutants perturb large-scale LBD dynamics, we computed conformational free energy landscapes for the apo state and glycine complexes of wild-type (WT) ML032222a and the R703K and E423S mutants, which weaken and break the interdomain salt bridge, respectively. This analysis reveals that, similar to vertebrate GluN1 and GluN3 glycine binding subunits, the apo state for ML032222a can access closed cleft conformations, although it is more stable in slightly open conformations. The R703K and E423S mutants destabilize closed cleft conformations for the glycine complex. Conformational dynamics inferred from the free energy landscapes suggest that the interdomain salt bridge is positioned at the most likely point of ligand entry to (and exit from) the binding pocket and thus acts as a steric barrier regulating the binding and dissociation of glycine.     

The IL-10 polarized cytokine pattern in innate and adaptive immunity cells contribute to the development of FVIII inhibitors

Background

Hemophilia A (HA) is an X-linked inherited bleeding disorder, resulting from a qualitative or quantitative deficiency of clotting factor VIII (FVIII). Antibodies against FVIII, also called inhibitors, block the procoagulant activity of FVIII; thus, impairing hemostatic activity in patients with HA. The exact mechanism underlying the immunological events behind the development of inhibitors remains unknown. This study aimed to understand immune response to FVIII in patients with HA who were either positive [HAα-FVIII(+)] or negative [HAα-FVIII(−)] for inhibitors.

Methods

Cytokine profiles [interferon-γ (IFN − γ), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), IL-5, and IL-10] of innate and adaptive immune cells present in the peripheral blood of participants were characterized.

Results

Presence of inhibitors was significantly associated with decreased frequencies of TNF-α-positive monocytes and neutrophils, IL-5-positive monocytes, IL-4-positive neutrophils, and increased frequencies of IL-10-positive neutrophils and T cells. T cells from HAα-FVIII(−) patients expressed increased levels of almost all cytokines. In contrast, HAα-FVIII(+) patients showed lower levels of all cytokines in CD4⁺ and CD8⁺ T cells, except IL-10. B cells from HAα-FVIII(−) patients expressed increased levels of IL-4 while those from HAα-FVIII(+) patients expressed increased levels of IL-10.

Conclusions

The global cytokine profiles of innate and adaptive immune cells showed an anti-inflammatory/regulatory pattern in HAα-FVIII(+) patients and a mixed pattern, with a bias toward inflammatory cytokine profile, in HAα-FVIII(−) patients. The occurrence of these profiles seems to be associated with presence FVIII inhibitors.     

Guidelines for ethical behavior relating to clinical practice issues in neuromuscular and electrodiagnostic medicine

The American Association of Neuromuscular & Electrodiagnostic Medicine (AANEM) developed guidelines to formalize the ethical standards that neuromuscular and electrodiagnostic (EDx) physicians should observe in their clinical and scientific activities. Neuromuscular and EDx medicine is a subspecialty of medicine that focuses on evaluation, diagnosis, and comprehensive medical management, including rehabilitation of individuals with neuromuscular disorders. Physicians working in this subspecialty focus on disorders of the motor unit, including muscle, neuromuscular junction, axon, plexus, nerve root, anterior horn cell, and the peripheral nerves (motor and sensory). The neuromuscular and EDx physician's goal is to diagnose and treat these conditions to mitigate their impact and improve the patient's quality of life. The guidelines are consistent with the Principles of Medical Ethics adopted by the American Medical Association and represent a revision of previous AANEM guidelines. Muscle Nerve 52 : 1122–1129, 2015